Filamentous fungi are used for the production of enzymes and proteins for a variety of industrial biotechnology applications. Of the fungi used for enzyme production, strains of Trichoderma reesei (the asexual anamorph of Hypocrea jecorina) are particularly prominent due to their ability to secret large amounts (>50 g/L) of useful enzymes in industrial fermentations. Different high-productivity (and “hyperproductive”) Trichoderma strain lineages have been developed by different groups, almost all originating from QM6a, the “wild-type” environmental strain (Bailey and Nevalainen, 1981, Enzyme Microb. Technol. 3: 153; Vitikainen et al., 2010, BMC Genomics 11: 441, and references therein). Reported examples of high-productivity strains include QM9414 (Mandels and Andreotti, 1978, Process Biochemistry 13: 6), Rut-C30 (Eveleigh and Montenecourt, 1979, Adv. Appl. Microbiol. 25: 57), CL847 (Durand et al., 1988, Enzyme Microb. Technol. 10: 341) and M2C38 (U.S. Pat. No. 6,015,703). High-productivity strains are used to express both enzymes endogenous to the organism as well as heterologously expressed and secreted proteins. Such enzymes and proteins include cellulases, hemicellulases, amylases, proteases, laccases and esterases—all of which have been expressed for commercial purposes. Efforts continue to identify factors that regulate protein productivity so that further improvements can be made (Le Crom et al., 2009, P.N.A.S. USA 106: 16151).
High productivity is dependent upon multiple factors. Depending upon the particular strain, the carbon source and feed rate will determine which genes for secreted enzymes are induced and/or repressed, as well as the growth and enzyme production rates observed. The carbon source acts through transcription factors and their cognate promoters that are either activated or repressed under certain feed conditions. Expression of proteins is typically driven by promoters for the major cellulases and hemicellulases (e.g., Cel6A, Cel7A, Xyn11B) or hybrids thereof, e.g., a hybrid of the Cel7A and Xyn11B promoter regions and secretion signals (U.S. Pat. No. 6,015,703). The transcription factors that regulate these promoters have been partially identified and characterized. Once transcribed and translation is initiated, secretion depends upon the particular signal sequence and the secretion apparatus of the cell, both of which have been manipulated to improve productivity. All steps from carbon source through secretion can be manipulated to improve productivity by identifying and altering the regulatory proteins and networks that control the cascade of functions.
Cellulase and hemicellulase expression can be controlled, in part, by the choice of carbon source (Ilmén et al., 1997, Appl. Environ. Microbiol. 63: 1298; Mach and Zeilinger, 2003, Appl. Microbiol. Biotechnol. 60: 515; Schmoll and Kubicek, 2003, Acta Microbiologica et Immunologica Hungarica 50: 125; Ahamed and Vermette, 2009, Bioresources Technology 100: 5979). Typically, cellulose and beta-linked gluco-oligosaccharides (e.g., cellobiose, sophorose, gentiobiose, laminaribiose, lactose) induce expression of cellulases and their accessory enzymes (Mandels et al., 1962, J. Bacteriology 83: 400; Sternberg and Mandels, 1979, J. Bacteriology 139:761; Foreman et al., 2003, J. Biol. Chem. 278: 31988). Similarly, xylan, xylose and xylo-oligosaccharides will induce hemicellulases and their accessory enzymes (Royer and Nakas, 1990, Appl. Environ. Microbiol. 56: 2535; Zeilinger et al., 1996, J. Biol. Chem. 271: 25624). Glucose represses (hemi)cellulase expression in cells with a functioning cre1 gene (Ilmen et al., 1996, Mol. Gen. Genet. 251: 451). Nitrogen has well documented effects on cellular reproduction and morphology (Steyaert et al., 2010a, Fungal Biology 114: 179; 2010b, Microbiology 156: 2887), which in turn effects large scale production results (Ahamed and Vermette, 2009, Bioresources Technology 100: 5979). Nitrogen may also play a role in cellulase production. For example, it has been reported that cellulase production is elevated in an A. niger strain containing a constitutively activated nitrogen regulator AreA, while cellulase production is reduced in an AreA loss-of-function mutant grown in cellulose-induced cultures using ammonium as a nitrogen source (Lockinton et al., 2002, Fungal Genet. Biol. 37: 190).
Several major transcription factors have been identified that interact with the promoter regions of cellulase and hemicellulase genes and regulate their expression (Mach and Zeilinger, 2003, Appl. Microbiol. Biotechnol. 60: 515; Schmoll and Kubicek, 2003, Acta Microbiologica et Immunologica Hungarica 50:1 25; Kubicek et al., 2009, Biotechnology for Biofuels 2:19 and references therein). Cre1 (catabolite repression) mediates carbon catabolite repression and blocks cellulase expression when the cells are grown on glucose and other non-inducing carbohydrates (Ilmen et al., 1996, Mol. Gen. Genet. 251: 451). The gene for Cre1 is often defective or deleted in high-productivity strains (Seidl et al., 2008, BMC Genomics 9:327; Nakari-Setälä et al., 2009, Appl. Environ. Microbiol. 75: 4853). However, recent data suggest that Cre1 may also have a role in upregulation by other factors involved in cellulase and hemicellulase expression (Portnoy et al., 2011, Eukaryotic Cell 10: 262). Xyr1 (xylanase regulator) is an essential transcriptional activator that promotes expression of cellulases and hemicellulases (Stricker et al., 2006, Eukaryotic Cell 5: 2128; Stricker et al., 2007, FEBS Letters 581: 3915; Stricker et al., 2008, Appl. Microbiol. Biotechnol. 78: 211). Ace1 (activator of cellulase expression) has been identified as a repressor of cellulases and xylanases (Aro et al., 2003, Appl. Environ. Microbiol. 69: 56). Ace2, in contrast, appears to be an activator of cellulase expression under certain conditions (Aro et al., 2001, J. Biol. Chem. 276: 24309). The Hap complex (heme activator protein, named after homologous proteins originally identified in Saccharomyces cerevisiae) has been shown to interact with regulatory regions in cellulase promoters that are necessary for expression (Zeilinger et al., 2001, Mol. Genet. Genom. 266:56). Strains of Trichoderma have been isolated that cannot be induced to produce cellulases at more than basal levels, presumably due to defects in one or more of these global regulators of cellulase expression (Nevalainen and Palva, 1978, Appl. Environ. Microbiol. 35: 11; Torigoi et al., 1996, Gene 173: 199).
While feed choice and the transcription factors associated with induction and repression will determine the levels of particular mRNAs transcribed, the feed rate and secretory capacity of a particular strain will determine how much carbon in the feed goes to secreted protein versus diversion into biomass, and how much and how long protein can be secreted from the cell before feedback signals cause secretion rates to diminish. Increasing feed rate will increase protein productivity up to a point past which productivity will decrease precipitously and feed will go primarily to making biomass (Pakula et al., 2005, Microbiology 151: 135). Strains fermented under conditions leading to high productivity may be limited by the capacity of the secretory pathway. Fungal cells respond naturally by increasing expression of secretory pathway components (Saloheimo et al., 1999, Mol. Gen. Genet. 262: 35; 2004, Appl. Environ. Microbiol. 70: 459). Improvement of secretion can be achieved by intentionally over-expressing genes involved in the secretory pathway (Kruszewska et al., 1999, Appl. Environ. Microbiol. 65: 2382; 2008, Acta Biochimica Polonica 55:447). Ultimately, secretion stress can result in misfolding of proteins in the secretory pathway which will activate the unfolded protein response (UPR) and in turn down-regulate the expression of secreted proteins (Saloheimo et al., 2003, Molecular Microbiology 47: 1149; Valkonen et al., 2004, Mol. Genet. Genom. 272:443). An additional response in Trichoderma—repression under secretion stress (RESS)—further down-regulates expression of secreted proteins (Pakula et al., 2003, Mol. Genet. Genom. 272:443).
Trichoderma isolates resulting from mutagenesis that are unable to produce cellulases have been reported in the literature. Torigoi et al. (1996) have previously characterized four cellulase deficient Trichoderma mutants (QM9136, QM9977, QM9978 and QM9979) obtained by UV irradiation of QM6a (Mandels et al., 1971). All of the described mutants fail produce detectable cel5A, cel6A, cel7A and cel7B transcripts when induced with sophorose or cellulose. Failure to produce cellulase in one of these mutants (QM9979) was linked to its inability to uptake cellulase di-saccharide inducer, and postulated through functional analysis to carry a defective/mutated β-glycoside permease (Kubicek et al., 1993). Still, identity of the specific gene involved and nature of mutation involved was not described
The present invention is based on the identification of a gene encoding a polypeptide involved in moderating the development of a heritable cellulase deficient phenotype that arises after prolonged fermentation of filamentous fungi. The identified gene also regulates the ability of the fungi to tolerate aggressive fermentation conditions that enable high productivity. Disclosed herein is a means for modifying or tailoring fungal cells to meet the specific requirements of different enzyme production conditions by decreasing or increasing the expression of the gene.